Preparation of nitrogen mustard derivatives

ABSTRACT

This invention relates to an improved synthesis of 5-(N-[(S)-N-{N,N-bis(2-chloroethyl)amino}phenoxycarbonyl)-γ-glutamyl]amino)isophthalic acid (also named ZD9063P), a prodrug used in Antibody Directed Enzyme Prodrug Therapy (ADEPT), a targeted cytotoxic cancer therapy. Another aspect of the invention comprises a compound of Formula (I) in which R 1  and R 2  are chloro, R 3  is an alphamethylbenzylamine salt of carboxylic acid, (R 4 ) n  represents a benzyl protected 3,5-dicarboxylic acid and the asterisked chiral carbon in Formula (I) has S configuration, preferably in crystalline form.

[0001] The present invention relates to an improved synthesis of5-(N-[(S)-N-{N,N-bis(2-chloroethyl)amino}phenoxycarbonyl)-γ-glutamyl]amino)isophthalicacid (also named ZD9063P herein), a prodrug used in Antibody DirectedEnzyme Prodrug Therapy (ADEPT), a targeted cytotoxic cancer therapy.

[0002] The use of cytotoxic drugs is limited by their general toxicityand a highly desirable aim is the targeted delivery of the activecytotoxic agent. A novel approach under development is the dosing of thepatient with an antibody/enzyme complex which binds specifically at thesite of the tumour. Subsequent treatment with a synthetic compound, theProdrug, which bears a masked cytotoxic entity and has been specificallydesigned to be cleaved by the enzyme, delivers the cytotoxic agent, theDrug, at the site of the tumour.

[0003] The known synthesis of ZD9063P involved a using a nitrogenmustard, N,N-bis(2-chloroethyl)aminophenol, and being the actualcytotoxic agent and would pose very significant containment problems ifused on a manufacturing scale (see U.S. Pat. No. 5,405,990; Example 46).Therefore there was a need for an improved synthetic route, particularlya route more suited to large scale manufacture.

[0004] The present invention is based on the discovery of a new,efficient and convergent route to ZD9063P and structurally relatedprodrugs by converting a readily available starting material to a keyintermediate suitable for both chemical and enantiomeric purification.The success of the synthetic approach arose from the use of a catalystto obtain regiospecific opening of an anhydride ring, incorporation of areaction at an elevated pressure to avoid a solvent exchange and thedevelopment of an improved practical procedure for the deprotection oft-butyl esters.

[0005] According to one aspect of the invention there is provided amethod of preparation of a compound of Formula I or an R⁴ deprotectedderivative thereof

[0006] wherein

[0007] R¹ and R² independently represent chloro, bromo, iodo or OSO₂Me;

[0008] R³ represents COOH or a salt of carboxylic acid;

[0009] n is 1,2, 3 or 4;

[0010] R⁴ represents a protected form of COOH, tetrazol-5-yl or SO₃H;and

[0011] in which the method comprises reacting a compound of Formula II

[0012] with a compound of Formula III

[0013] to give a compound of Formula I and optionally further comprisingdeprotecting R⁴ and optionally converting the product thus obtained intoa pharmaceutically acceptable salt thereof.

[0014] R¹ and R² are preferably chloro. R³ is preferably analphamethylbenzylamine salt of carboxylic acid. R⁴ is preferably aprotected form of COOH, especially COOBn. Preferably n is 2 andespecially a 3,5 dicarboxylic acid in protected form is preferred.Preferably the asterisked chiral carbon in Formula I has Sconfiguration.

[0015] Preferably the reaction is performed in the presence of a highlynucleophilic but weakly basic amine. Preferably the amine is DMAP(4-dimethylaminopyridine) or 4-pyrrolidinopyridine, especially DMAP.This has the advantage of regioselective opening of the cyclic anhydridering.

[0016] Preferably the reaction is performed at a temperature of −50° to30°, more preferably −40 to 0°, more preferably −40 to −10° andespecially at about −35 to −25°. Preferred temperature ranges have theadvantage of giving improved yield of desired reaction products.

[0017] According to another aspect of the invention there is provided amethod of preparation of a a compound of Formula II which comprisesremoving the tBu protecting groups from a compound of Formula IV

[0018] in the presence of methane sulphonic acid followed by cyclisationto the anhydride to give a compound of Formula II. This is advantageousbecause the de-esterification could be readily achieved by the extendedreaction of trifluoroacetic acid but a significant practical problemencountered was the complete removal of the excess trifluoroacetic acidprior to the activation step. A large excess of trifluoroacetic acid isrequired to displace the equilibrium set up between its t-butyl esterand the corresponding t-butyl ester of the substrate. Complete reactionis normally only achieved by multiple treatments removing volatilesubstances by distillation, a time-consuming procedure for large scaleoperation. The t-butyl ester of methanesulphonic acid is unstable and sothe corresponding equilibrium can be displaced by the formation ofisobutylene which escapes from the system.

[0019] According to another aspect of the invention there is provided amethod of preparation of a compound of Formula IV in which R¹ and R² arechloro which comprises reacting a compound of Formula V

[0020] with methane sulphonyl chloride in the presence of methylenechloride in a pressure vessel to give a compound of Formula IV in whichR¹ and R² are chloro. This is advantageous for the following reasons.The readily available starting material reacts with methanesulphonylchloride/diisopropylethylamine in methylene chloride solution to give anessentially quantitative yield of the corresponding dimesylate. Themesylate groups (also termed “Ms” or “methanesulphonyl” herein) aredisplaced by the chloride ion present but the reaction is very slow evenat reflux. An alternative solvent is not an attractive option asmethylene chloride is the most suitable solvent for obtaining an initialsolution of starting material, a necessary requisite for a high chemicalconversion to the dimesylate. The solvent used must be compatible withthe strongly acidic reaction conditions required for removal of thet-butyl groups as the isolation of 10 (for numerically identifiedcompounds, see Schemes below) as a crystalline solid is not easy and itis more convenient to proceed directly with the deprotection stage. Inaddition, both 10 and 14 contain the nitrogen mustard system, albeit ina less activated form, and handling of such intermediates would causeconcern on a production scale. The problem of the slow rate of thedisplacement reaction at reflux in methylene chloride solution (40° C.)was overcome, without introducing a change to a higher boiling solvent,by operating the reaction under pressure. Complete exchange occurs after18 hours at 75° C. and the pressure generated in the system, about 2BarG, is entirely consistent with operation in a standard productionplant.

[0021] According to another aspect of the invention there is provided acompound of Formula I in which R¹ and R² are chloro, R³ is analphamethylbenzylamine salt of carboxylic acid and (R⁴)_(n) represents abenzyl protected 3,5-dicarboxylic acid. Preferably the compound is incrystalline form.

[0022] Another aspect of the invention comprises a compound of Formula I

[0023] in which R¹ and R² are chloro, R³ is an alphamethylbenzylaminesalt of carboxylic acid, (R⁴)_(n) represents a benzyl protected3,5-dicarboxylic acid and the asterisked chiral carbon in Formula I hasS configuration. Preferably the compound is in crystalline form whereinsaid alphamethylbenzylamine is in enantiomerically pure (R)- or(S)-configuration.

[0024] The crystalline form of this compound gives the advantage of easeof handling during manufacture, particularly at large scale.

[0025] Explanation of the discovery of the advantageous synthetic routeis set out below. A molecule 8, possessing the same basic aryl urethanederivative of (S)-glutamic acid as ZD9063P, was available. (Scheme 1)

[0026] The two key transformations required are the conversion of thehydroxyl groups to chloro groups and the regioselective coupling of thedibenzyl ester of 5-aminoisophthalic acid with the glutamic acid residuegiving a compound 9 which can be converted to ZD9063P by catalytichydrogenation. The key decision is the order in which the chlorinationreaction and the introduction of the dibenzyl ester of5-aminoisophthalic acid moiety are carried out. (Scheme 2)

[0027] Initially, the partial hydrolyses of 8 and of itsdichloro-analogue 10 were investigated to provide differentiallyprotected derivatives of glutamic acid. Trifluoroacetic acid selectivelycleaved the less hindered ester group but it was not possible to obtainsolution yields of the desired regioisomer of greater than about 50%because further de-esterification continually occurred.^(i)

[0028] The physical characteristics of the chlorinated species as wellas the desire to avoid the conversion of the hydroxyl group to thechloro group at a late stage in the synthesis suggested that a dichloroderivative should be chosen as the key starting material for couplingwith the dibenzyl ester of 5-aminoisophthalic acid. In addition,approaches based on the opening of the corresponding cyclic anhydridewere considered to be more selective than activation of the acyclicsystem with, for example, a chloroformate ester.

[0029] Control of the regioselective opening of anhydrides of glutamicacid derivatives by methanol in the presence of triethylamine containingvarying amounts of DMAP has been described. The ratio of regioisomers isreversed from about 1:7 to about 7:1 α/γ by the addition of DMAP.^(ii)

[0030] Regioselective opening of the anhydrides of phthaloyl derivativesof glutamic acid with amines has been described leading to the γ-isomerand specific reference was made to the opening of the anhydrides ofurethane derivatives of the anhydride of glutamic acid with ammonialeading to predominantly the α-isomer.^(iii) The regioselectivity ofreaction with amines has also been controlled in urethane derivatives ofglutamic and aspartic acid anhydrides by choice of reactionsolvent.^(iv v vi) No examples in which DMAP affected theregioselectivity of nucleophilic attack by amines on derivatives of thecyclic anhydrides of glutamic acid have been found.

[0031] The invention will now be illustrated by the followingnon-limiting Examples in which:

[0032] Scheme 1 shows synthesis of compound 8 in which a=TMS-Cl,b=4-nitrophenol chloroformate, c=esterification, d=reduction,e=hydroxyethylation

[0033] Scheme 2 shows options for key transformations in which the boldarrows show the route developed with key intermediates and the dottedarrows show alternative route options with possible intermediates

[0034] Scheme 3 shows a model experiment

[0035] Scheme 4 shows a new synthetic route to ZD9063P in whicha=MsCl/Hunig's base, b=heat

[0036] Reagents were purchased from standard suppliers.

[0037] NMR spectra were run at 270 MHz (proton) and at 67.7 MHz (carbon)in d₆-DMSO or d₆-DMSO/TFA solution and are reported in parts per milliondown field from internal TMS. The signals assigned to TFA (159.0, q,J=60.9 Hz, 115.3, q, J=440 Hz) are omitted from the description of the¹³C spectrum for each compound. HPLC analyses were conducted using aHiChrome™ RPB column, solvent system acetonitrile/water/TFA 640/360/1(v/v/v), flow rate 1 or 2 mL/min and detection at 254λ

EXAMPLE 1 Model Studies

[0038] Derivatives of glutamic acid were used as mechanistic probes toinvestigate the reaction of anhydrides with the dibenzylester of5-aminoisophthalic acid.

[0039] CBZ-Glutamic acid (CBZ=benzyloxycarbonyl) was converted by DCC(DCC or DCCI=dicyclohexylcarbodiimide) to its cyclic anhydride which wasreacted with the p-toluenesulphonate salt of the dibenzylester of5-aminoisophthalic acid in the presence of an excess of a tertiaryamine. Two products were seen in the reactions involving triethylamineand 4-methylmorpholine whereas, completely unexpectedly, a singleproduct resulted when DMAP was used as the base. The compounds wereisolated and shown to be 11 and 12, the regioisomers from the opening ofthe anhydride ring system.^(vii) (Scheme 3)

[0040] The single compound formed in the DMAP catalysed reactioncorresponded to the more polar regioisomer 12 which possesses theZD9063P substitution pattern. This key observation means that the basiccarbon skeleton of ZD9063P can be assembled without the need for anexpensive, differentially protected, glutamic acid derivative.

[0041] Racemisation of the chiral centre of the glutamie acid residue isa possible problem with the base catalysed opening of the anhydridering. The proposed reaction was modelled using the(−)-menthyloxycarbonyl derivative of (R)-glutamic acid because anenantiomeric analytical method for 9 was not initially available. Thecorresponding anhydride was reacted with the dibenzylester of5-aminoisophthalic acid in the presence of various tertiary amines andthe following results obtained.^(viii ix)

[0042] Results of Opening the Anhydride under Various Conditions (%Yields) Glutamic acid Glutamic acid Desired enantiomer of Regio-enantiomer of Base product desired product isomer regioisomer4-Methylmorpholine 50 0 50 0 Pyridine 50 0 50 0 Triethylamine 25 25 2525 No additional base 15 0 85 4-Pyrrolidino- 82 16 2 0 pyridine DMAP (at+20° C.) 88 8 4 0 DMAP (at −30° C.) 96 2 2 0

[0043] The results demonstrate that a highly nucleophilic but weaklybasic amine possesses the desired catalytic activity for the desiredregioselective opening of the cyclic anhydride of a glutamic acidderivative by the dibenzyl ester of 5-aminoisophthalic acid.

EXAMPLE 2 Development of the Manufacturing Route

[0044] With the information from the modelling studies, the conversionof 8 to the desired cyclic anhydride was then reinvestigated with a highdegree of confidence that the approach would ultimately lead to apractical manufacturing process for ZD9063P. (Scheme 4)

[0045] The readily available starting material 8 reacts withmethanesulphonyl chloride/diisopropylethylamine in methylene chloridesolution to give an essentially quantitative yield of the correspondingdimesylate. The mesylate groups are displaced by the chloride ionpresent to give 10 but the reaction is very slow even at reflux. Analternative solvent is not an attractive option as methylene chloride isthe most suitable solvent for obtaining an initial solution of 8, anecessary requisite for a high chemical conversion to the dimesylate.The solvent used must be compatible with the strongly acidic reactionconditions required for removal of the t-butyl groups as the isolationof 10 as a crystalline solid is not easy and it is more convenient toproceed directly with the deprotection stage. In addition, both 10 and14 contain the nitrogen mustard system, albeit in a less activated form,and handling of such intermediates would cause concern on a productionscale.

[0046] The problem of the slow rate of the displacement reaction atreflux in methylene chloride solution (40° C.) was overcome, withoutintroducing a change to a higher boiling solvent, by operating thereaction under pressure. Complete exchange occurs after 18 hours at 75°C. and the pressure generated in the system, about 2 BarG, is entirelyconsistent with operation in a standard production plant.

[0047] The de-esterification of 10 could be readily achieved by theextended reaction of trifluoroacetic acid but a significant practicalproblem encountered was the complete removal of the excesstrifluoroacetic acid prior to the activation step. A large excess oftrifluoroacetic acid is required to displace the equilibrium set upbetween its t-butyl ester and the corresponding t-butyl ester of thesubstrate. Complete reaction is normally only achieved by multipletreatments removing volatile substances by distillation, atime-consuming procedure for large scale operation. The t-butyl ester ofmethanesulphonic acid is unstable and so the corresponding equilibriumcan be displaced by the formation of isobutylene which escapes from thesystem.^(x) In this way, complete deprotection of 10 to 14 is achievedusing only 0.75 mole equivalents of methane sulphonic acid (18 h at 40°C.). Practical problems arose because the product precipitated as anoily solid in the presence of methane sulphonic acid. However,neutralisation of the added methane sulphonic acid with an equivalent ofDMAP successfully overcame the problem giving a solution with a ‘pH’suitable for cyclisation to the anhydride with DCC.

[0048] The corresponding anhydride 15 of 14 reacts at −30° C. with thedibenzylester of 5-aminoisophthalic acid in the presence of oneequivalent of DMAP. The reaction is largely complete within twohours.^(xi)

[0049] Although it is possible to isolate the free acid form of 9 fromthe reaction mixture after work-up by crystallisation of the evaporatedresidue from an ethyl acetate/cyclohexane solvent mixture, it wassimpler to isolate the Salt 16 formed with (S)-α-methylbenzylaminedirectly from a mixture of methylene chloride and acetonitrile.Formation of the salt gave considerable purification as well as anopportunity for any necessary subsequent enantiomeric enrichment.^(xii)

[0050] The structure of 16 has been correlated with ZD9063P byconversion to the corresponding free acid followed by removal of thebenzyl groups by catalytic hydrogenation. The resulting tri-acid wasidentical to an authentic sample of ZD9063P by ¹H and ¹³C NMR andHPLC.^(xiii)

[0051] In conclusion, we have developed a new, efficient and convergentroute to ZD9063P by converting a readily available starting material toa key intermediate suitable for both chemical and enantiomericpurification. The success of the synthetic approach arose from the useof a catalyst to obtain regiospecific opening of an anhydride ring,incorporation of a reaction at an elevated pressure to avoid a solventexchange and the development of an improved practical procedure for thedeprotection of t-butyl esters

EXAMPLE 3 (S)-(α)-Methylbenzylamine salt of the dibenzyl ester of5-(N-[(S)-N-{N,N-bis(2-chloroethyl)amino}phenoxycarbonyl)-γ-glutamyl]amino)isophthalicacid. (16)

[0052] A solution of 8 (193 g, 0.40 mole) and N,N-diisopropylethylamine(124 g, 167 mL, 0.96 mole) in dichloromethane (1.5 L) was protected fromatmospheric moisture and stirred at 0° C. Methane sulphonyl chloride(101 g, 68 mL, 0.878 mole) was added at such a rate so that the reactiontemperature remained between 0-5° C. A wash of methylene chloride (200mL) was added via the dropping funnel. Analysis by HPLC showed greaterthan 97% AN conversion to the corresponding dimesylate after 2 h.^(xiv)The solution was transferred to an autoclave, methylene chloride (460mL) added and the solution agitated and heated in a sealed vessel for 18h (jacket temperature 75° C., pressure generated 1.6 BarG). Analysis byHPLC showed a 97% AN conversion to 10. A sample was isolated bychromatography for characterisation.

[0053]¹H NMR 7.95 (d, J=10 Hz, 1H), 6.94, (d, J=10 Hz, 2H), 6.72 (d,J=10 Hz, 2H), 4.0-3.9, (m, 1H), 3.73 (s, 8H), 2.4-2.3 (m, 2H), 2.0-1.9(m,1H), 1.9-1.75 (m, 1H), 1.42 (s, 18H)

[0054] The cooled reaction mixture was washed with water (570 mL),aqueous citric acid solution (570 mL of 20% w/v) and water (570 mL). Thefinal two separations emulsified slightly. The solution was passedthrough Whatman 1PS™ filter paper to remove extraneous water.Methanesulphonic acid (57.7 g, 38.9 mL, 0.60 mol) was added and thesolution stirred and distilled and one litre of distillate collected.Methylene chloride (1.0 L) was added, the mixture heated and one litreof distillate collected. Further methylene chloride (0.7 L) was addedand the mixture heated at reflux for 18 h to give an oily mixture.Analysis of the supernatant solution showed the absence of significantquantities of 13 and any intermediate mono esters. A solution of DMAP(70.8 g, 0.58 mol) in methylene chloride (200 mL) was added slowly. Theoil dissolved to give a dark solution and analysis by HPLC showed >95%AN conversion to the desired product, 14, present as a salt with DMAP.

[0055] The methylene chloride solution of 14 was inerted by a stream ofnitrogen and a solution of DCCI (88.3 g, 0.428 mol) in methylenechloride (220 mL) added over about 1 h maintaining a temperature of5-10° C. After a further hour, analysis by HPLC showed essentiallycomplete conversion of 14 to the corresponding anhydride 15. Thesolution was cooled to −50° C. and a solution of DMAP (48.8 g, 0.40 mol)in methylene chloride 200 mL) followed by a solution of thedibenzylester of 5-aminoisophthalic acid (144 g, 0.40 mol) in methylenechloride(800 mL), the temperature being maintained throughout at −50° C.Periodic analysis of the reaction mixture showed it to be essentiallycomplete after 3 h and it was allowed to reach ambient temperature overnight. Water (2.5 L) was added, the mixture stirred for 1 h, filtered toremove precipitated N,N¹-dicyclohexylurea and the phases separated. Theorganic phase was washed with aqueous citric acid (2 L of 10% w/v) andwater (2 L). The organic solution was filtered through Whatman 1PS™filter paper to remove adventitious water and divided into two portionsof 1.38 L.

[0056] One portion was distilled to half volume at atmospheric pressure,diluted with acetonitrile (1.3 L) and stirred at 40° C. whilst asolution of (S)-α-methyl benzylamine (24.2 g, 0.20 mol) in acetonitrile(80 mL) was added. Crystallisation commenced quickly and the slurry wasallowed to cool to ambient temperature over 2-3 h. The product wasfiltered, washed with (2×100 mL methylene chloride/acetonitrile 1/2 v/v)and dried at 30° C. The weight of product 16 was 134 g, 0.154 mol.(76.9% from 8).

EXAMPLE 4 Recrystallisation of the (α)-Methylbenzylamine salt of thedibenzyl ester of5-(N-[(S)-N-{N,N-bis(2-chloroethyl)amino}phenoxycarbonyl)-γ-glutamyl]amino)isophthalicacid. (16)

[0057] The salt 16 (20.0 g, 22.9 mmol) was dissolved in acetonitrile(700 mL) at reflux and cooled to 20° C. The crystalline product wasfiltered, washed with acetonitrile (100 mL) and dried at 50° C. Yield14.6 g, 16.7 mmole (73.0% yield)

[0058]¹H NMR 10.68 (s, 1H), 8.54 (s, 2H), 8.18 (s, 1H), 7.90 (d, J=10Hz, 1H), 7.52-7.23 (m, 15H), 6.90 (d, J=10 Hz, 2H), 6.68 (d, J=10 Hz,2H), 5.38 (s, 4H), 4.28 (q, J=10 Hz, 1H), 3.85-3.75 (m, 1H), 3.70 (s,8H), 2.48-2.37 (m, 2H), 2.20-2.00 (m, 1H), 2.00-1.98 (m, 1H), 1.44 (d,J=10 Hz, 3H)

[0059]¹³C 173.8, 171.5, 165.1, 155.2, 144.1, 142.5, 140.6, 139.5, 136.2,130.8, 129.0, 128.9, 128.8, 128.5, 128.4, 127.0, 124.5, 124.0, 122.9,67.0, 53.8, 52.8, 50.5, 41.2, 33.1, 26.7, 20.8 Calc for C₄₆H₄₈N₄O₉Cl₂:C, 63.37; H, 5.55; N, 6.43, Cl, 8.13. Found: C, 63.34; H, 5.52; N, 6.41,Cl, 8.13.

[0060] Quantitative analysis by ¹H NMR showed the strength to be 100%relative to an internal standard of maleic acid. Additionally, althoughquantitative analysis by HPLC showed that recrystallisation increasedthe strength of the product by only 1-2%, comparison of the ¹H spectrashowed that low levels of structurally unrelated aliphatic componentshad been removed.

EXAMPLE 5 Dibenzyl ester of5-(N-[(S)-N-{N,N-bis(2-chloroethyl)amino}phenoxycarbonyl)-γ-glutamyl]amino)isophthalicacid. (9)

[0061] The above salt 16 (5.0 g, 5.74 mmol) was partitioned betweenethyl acetate (150 mL) and an aqueous solution of citric acid (100 mL of20% w/v). The resulting organic phase was re-washed with an aqueoussolution of citric acid (50 mL of 20% w/v) followed by water (3×50 mL).The organic phase was filtered through Whatman 1PS™ filter paper and thesolvent removed under reduced pressure to give the desired product 9 asan amorphous foam. Yield 4.0 g, 5.34 mmol (93% yield)

[0062]¹H NMR 10.5, (s, 1H), 8.60, (s, 2H), 8.28 (s, 1H), 8.00 (d, J=10Hz, 1H), 7.5-7.3 (m, 10H), 6.94, (d, J=10 Hz, 2H), 6.72 (J=10 Hz, 2H),5.38 (s, 4H), 4.2-4.1, (m, 1H), 3.73 (s, 8H), 2.68-2.50 (m, coincideswith signal from d⁶-DMSO) 2.62-2.50 (m, 2H), 2.32-2.15 (m, 1H), 2.1-1.9(m, 1H)

[0063]¹³C NMR 173.9, 171.8, 165.1, 155.5, 144.1, 142.5, 140.8, 136.1,131.2, 128.8, 128.5, 128.2, 124.7, 124.4, 123.0, 112.6, 67.0, 53.8,53.0, 41.6, 33.0, 27.0

[0064] Analysis showed the presence of less than 1% of the(R)-enantiomer.^(xv)

EXAMPLE 65-(N-[(S)-N-{N,N-bis(2-chloroethyl)amino}phenoxycarbonyl)-γ-glutamyl]amino)isophthalicacid. (ZD9063P)

[0065] A sample of 10% palladium/carbon (200 mg) was slurry washed withredistilled THF (20 mL) and transferred to a flask. A solution of 9(0.30 g, 0.4 mmol) in redistilled THF (50 mL) was added. The slurry washydrogenated at ambient temperature and pressure. After 18 hours, whenremoval of the protecting groups was complete, the slurry was dilutedwith redistilled THF (10 mL) and the catalyst removed by filtration. Thesolvents were removed under vacuum to leave the product as an amorphoussolid, Yield 0.18 g, 0.315 mmol (78.9% yield)

[0066]¹H NMR^(xvi) 10.40, (s, 1H), 8.50, (s, 2H), 8.20, (s, 1H), 8.00(d, J=10 Hz, 1H), 7.00 (d, J=10 Hz, 2H), 6.75 (d, J=10 Hz, 2H),4.23-4.08 (m, 1H), 3.73, (s, 8H), 2.65-2.52 (m) overlaps with thed₆-DMSO signal, 2.35, (m, 1H), 2.10-1.95, (m,1H)

[0067]¹³C NMR 173.9, 171.2, 167.2, 155.3, 144.1, 142.6, 140.2, 132.2,125.2, 124.2, 122.8, 113.0, 54.2, 53.3, 41.7, 33.2, 27.2

[0068]^(i) The structure of the major regioisomer of the mixture wasassigned by comparison of the chemical shifts in the NMR spectra of theprotons adjacent to the carbonyl groups in the glutamic acid residue inthe acid and salt forms of the product. Formation of the ammonium saltresulted in an up-field shift of approximately 0.2 ppm in the positionof the methylene protons thus showing them to be adjacent to acarboxylic acid group and not an ester function.

[0069]^(ii) Jouin, P.; Castro, B.; Zeggaf, C.; Pantaloni, A.; Senet, J.P.; Lecolier, S.; Sennyey, G, Tetrahedron Lett. 1987, 28, 1665

[0070]^(iii) Sheehan, J. C.; Bolhofer, W. A.; J. Am. Chem. Soc 1950, 72,2469

[0071]^(iv) Cristea, I.; Mager, S.; Batiu, C.; Plé, G. Rev. Roum. Chim.,1994, 39(12), 1435

[0072]^(v) Huang X.; Luo X.; Roupioz Y.; Keillor J. W. J. Org. Chem.1997, 62, 8821

[0073]^(vi) Ksander G. M.;Yuan A. M.; Diefenbacher C. G.; Stanton J. L.J. Med. Chem. 1985, 28, 1606

[0074]^(vii) The key feature of the structural assignment was acomparison of the NMR spectra with that of CBZ-glutamic acid. Thechemical shift of the methine proton in the less polar compound wassignificantly different from the methine proton in CBZ-glutamic acidwhereas in the more polar compound the significant difference was in themethylene protons.

[0075]^(viii) It was not possible to analyse (HPLC or NMR) the anhydridefor the presence of the diastereomer to demonstrate the absence ofracemisation during the DCC mediated ring closure reaction.

[0076]^(ix) The anhydride of menthyloxycarbonyl-(S)-glutamic acid wassubjected to a similar series of reactions. Comparison with the similarproducts from the enantiomeric acid allowed the four possible productsto be assigned unambiguously on the HPLC trace.

[0077]^(x) King J. F.; du Manoir J. R.; J. Am. Chem. Soc. 1975 97 2566

[0078]^(xi) Two minor by-products are observed.

[0079]^(xii) A crystalline product was also obtained in about the sameisolated yield with (R)-α-methylbenzylamine. It was noticed that the twodiastereomeric salts did crystallise at significantly different ratesfrom the same solvent mixture but it is not known which salt would bemore useful in further work on enhancing the purity of the derivedZD9063P.

[0080]^(xiii) A sample of the regioisomeric Dibenzyl Ester was isolatedby chromatography and its structure confirmed by NMR. Removal of thebenzyl ester groups gave a compound very closely related to, but clearlydifferent from, ZD9063P by HPLC and NMR analysis.

[0081]^(xiv) AN—area normalised

[0082]^(xv) Analysis by chiral HPLC using a CHI-DMB column, solventsystem iso-hexane/isopropanol/TFA (75/25/0.1), flow 1 mL/min anddetection at 254λ.

[0083]^(xvixvi) ^(xvi) Residual toluene and THF were detected in bothspectra.

1. A method of preparation of a compound of Formula I or an R⁴deprotected derivative thereof

wherein R¹ and R² independently represent chloro, bromo, iodo or OSO₂Me;R³ represents COOH or a salt of carboxylic acid; n is 1,2, 3 or 4; R⁴represents a protected form of COOH, tetrazol-5-yl or SO₃H; and in whichthe method comprises reacting a compound of Formula II

with a compound of Formula III

to give a compound of Formula I and optionally further comprisingdeprotecting R⁴ and optionally converting the product thus obtained intoa pharmaceutically acceptable salt thereof.
 2. A method according toclaim 1 in which R¹ and R² are chloro, R³ is a alphamethylbenzylaminesalt of carboxylic acid, n is 2 and R⁴ is a protected form of COOH.
 3. Amethod according to claim 2 wherein R⁴ is COOBn.
 4. A method accordingto claim 3 wherein R⁴ is attached to the 3 and 5 positions of the phenylring.
 5. A method according to claim 4 wherein the asterisked chiralcarbon in Formula I has S configuration.
 6. A method according to anyone claims 1-5 wherein the reaction is performed in the presence ofDMAP(4-dimethylaminopyridine).
 7. A method according to claim 6 whereinthe reaction is performed at a temperature of −40 to 0°.
 8. A methodaccording to claim 7 wherein the reaction is performed at a temperatureof −35 to −25°.
 9. A method of preparation of a compound of Formula II

wherein R¹ and R² independently represent chloro, bromo, iodo or OSO₂Me;which comprises removing the tBu protecting groups from a compound ofFormula IV

in the presence of methane sulphonic acid followed by cyclisation to theanhydride to give a compound of Formula II.
 10. A method of preparationof a compound of Formula IV

in which R¹ and R² are chloro which comprises reacting a compound ofFormula V

with methane sulphonyl chloride in the presence of methylene chloride ina pressure vessel to give a compound of Formula IV in which R¹ and R²are chloro.
 11. A compound of Formula I

in which R¹ and R² are chloro, R³ is an alphamethylbenzylamine salt ofcarboxylic acid, (R⁴)_(n) represents a benzyl protected 3,5-dicarboxylicacid and the asterisked chiral carbon in Formula I has S configuration.12. A compound according to claim 11 in crystalline form wherein saidalphamethylbenzylamine is in enantiomerically pure (R)- or(S)-configuration.